Hemostasis is the process of arresting the outflow of blood from an injured blood vessel. For mammals, as well as many other organisms, the hemostatic process is critically important for continued survival. Defects in the hemostatic process can result in, for example, the inability to effectively form blood clots that serve to stop the loss of blood following vascular injury. In humans, individuals who suffer from an inability to form blood clots are called hemophiliacs. Of particular concern for hemophiliacs is the life-threatening risk that once started, bleeding will never cease.
Generally, hemophiliacs lack the ability to produce effective amounts of one or more substances ultimately required for the transformation of soluble fibrinogen into insoluble fibrin. For example, hemophiliacs who suffer from hemophilia B (also called “congenital factor IX deficiency” and “Christmas disease”) have an inability to produce effective levels of Factor IX. Factor IX is a key component of one of several “cascades” of reactions that result in the formation of blood clots. Critical for the cascade of reactions referred to as the “intrinsic pathway,” Factor IX ultimately influences the conversion of fibrinogen into the major component of blood clots, fibrin.
Although the process by which blood clots are formed is relatively complex, the role of Factor IX in the intrinsic pathway can be described briefly. When blood comes into contact with negatively charged surfaces and/or subendothelial connective tissues (as a result of, for example, tissue damage associated with a laceration), Factor XII (or Hageman factor) in the presence of other substances is transformed into Factor XIIa. Factor XIIa (along with other substances) transforms Factor XI into Factor XIa. In turn, Factor XIa (along with other substances) transforms Factor IX into Factor IXa. Factor VIII, Factor IXa, calcium ions and phospholipid micelles form a lipoprotein complex with Factor X and activate it to form Factor Xa. Thereafter, Factor Xa (along with other substances) converts prothrombin into thrombin, with the result that a relatively large amount of thrombin is produced over time. Relatively large amounts of thrombin convert fibrinogen into fibrin. Fibrin, in turn, forms the matrix or lattice responsible for the formation of blood clots. Factor IX's role in the intrinsic pathway of blood clotting is shown schematically below.

Affecting one out of 34,500 males, hemophilia B can result from any one of a variety of mutations of the Factor IX gene, which is located on the X-chromosome. Depending on the particular mutation, hemophilia B can manifest itself as severe, moderate or mild. Individuals suffering from the severest forms of hemophilia B entirely lack the ability to express active forms of Factor IX. Clinically, individuals affected with hemophilia B suffer from nose bleeds, easy bruising, joint hemorrhage, and prolonged bleeding from wounds. Current treatment of hemophilia B involves the infusion of exogenous Factor IX concentrate collected from human plasma or prepared via recombinant DNA techniques. Because these treatments serve only to supplement the lack of effective levels of Factor IX, individuals suffering from severe forms of hemophilia B require regular injections (as often as three times a week) of Factor IX concentrate throughout their lives. Patients suffering from even more moderate forms of hemophilia B often require injection of Factor IX concentrate before and/or following surgery and dental work.
Several commercial forms of Factor IX concentrates are available to provide replacement therapy for patients suffering from hemophilia B. For example, blood-derived Factor IX complex products (containing other factors) are sold under the BEBULIN VH® (Baxter Healthcare, Vienna, Austria), KONYNE 80® (Bayer Corporation, Eikhart Ind.), PROFILNINE SD™ (Alpha Therapeutic Corporation, Los Angeles Calif.), and PROPLEX T® (Baxter Healthcare, Glendale Calif.) brands. Somewhat more purified forms of Factor IX products are sold under the ALPHANINE SD® (Alpha Therapeutic Corporation, Los Angeles Calif.) and MONONINE® (Aventis Behring, Kankakee Ill.) brands. With respect to recombinantly prepared Factor IX concentrates, one product is currently available under the BENEFIX® (Wyeth/Genetics Institute, Cambridge Mass.) brand.
Generally, the recombinant source of Factor IX concentrate is favored over blood-derived sources since the latter involves the risk of transmitting viruses and/or other diseases. In addition, purity is often higher with the recombinant source, thereby avoiding potential problems arising from administering unwanted blood factors and other proteins generally present in blood-derived sources.
Notwithstanding the benefits of administering a recombinant-based formulation, the processing of recombinant-based products often requires the presence of certain proteins such as albumin, which can be present in the final formulation administered to the patient. As a result, patients who receive such formulations develop allergic reactions to these foreign proteins. In any event, both blood-derived and recombinant-based products suffer from the disadvantage of repeated administration.
PEGylation, or the attachment of a poly(ethylene glycol) derivative to a protein, has been described as a means to reduce immunogenicity as well as a means to prolong a protein's in vivo half-life. With respect to Factor IX, however, previous approaches for forming protein-polymer conjugates suffered from several deficiencies.
For example, U.S. Pat. No. 5,969,040 describes a process comprising the step of oxidizing vicinal diols of carbohydrate moieties in the activation region of Factor IX to form aldehydes. Following the oxidizing step, the described process includes the step of covalently attaching one or more non-antigenic polymers [such as a hydrazine-bearing poly(ethylene glycol) derivative] to the oxidized carbohydrate moieties. A problem with this approach, however, is the increased complexity attributed to the additional steps required to obtain an oxidized form of Factor IX. In addition, any oxidizers that may remain following the oxidation step may degrade the polymer associated with the conjugate. Finally, this approach is limited to conjugation using specific polymers (i.e., hydrazide-containing polymers) and specific regions on Factor IX (i.e., vicinal diols of carbohydrate moieties in the activation region of Factor IX).
The presence of oxidizers (present either as a result of the process described in U.S. Pat. No. 5,969,040, or from other causes) introduces additional challenges with respect to providing an acceptable pharmaceutical product of a polymer conjugated to Factor IX. Specifically, methionine and other hydroxyl-containing amino acids may be subject to unwanted oxidation in the presence of oxidizers, thereby introducing aldehyde groups. Any residual aldehydes not reacted with the polymeric reagent will be reactive and can potentially damage the protein. In order to address this problem, unreacted aldehydes need to be capped with glycine or other small molecule to stabilize the protein. In doing so, however, an analytical problem arises in that for regulatory purposes, a product should be readily defined; the introduction of additional components can frustrate otherwise straightforward product definition. In particular, the use of capping agents would present a particularly difficult challenge.
U.S. Pat. No. 6,037,452 describes attachment of a poly(alkylene oxide) to Factor IX, wherein attachment to Factor IX is effected through a poly(alkylene oxide) bearing one of the following reactive groups: triazine, acetyl, hydrazine, diazonium, amino, and succinimidyl ester. Again, however, the reference lacks disclosure of effecting attachment through polymers bearing reactive groups other than triazine, acetyl, hydrazine, diazonium, amino, or succinimidyl ester.
Thus, there remains a need in the art to provide additional conjugates between water-soluble polymers and moieties having Factor IX activity. In particular, there is a need to provide more simple processes for conjugating a polymer to a moiety having Factor IX activity. The present invention is therefore directed to such conjugates as well as compositions comprising the conjugates and related methods as described herein, which are believed to be new and completely unsuggested by the art.